Understanding the genetic diversity at molecular level is a prerequisite in developing strategies for effective conservation and utilization of chicken genetic resources. We studied the genetic variation within and between Bangladeshi native (Naked Neck, Frizzle and Non-descriptive indigenous) and exotic (White Leghorn, Rhode Island Red, Commercial layer and broiler) chicken populations by Random Amplified Polymorphic DNA (RAPD). Four out of the 20 random primers exhibited sufficient variability for studied populations. The four primers yielded a total of 39 distinct bands, 25 of which were polymorphic. Estimation of polymorphic loci, intra-population similarity indices and Neis gene diversity suggested that genetic diversities within a population were high in non-descriptive, Frizzle, Naked Neck, Rhode Island Red and White Leghorn chicken populations compared to the commercial layer and broiler populations. The coefficient of gene differentiation (GST = 0.34) and gene flow (Nm = 0.98) values reflected a high level of population differences. UPGMA dendrogram segregated the chicken populations in various degree based on their genetic distance. The overall genetic distance among native chicken was relatively low comparison to the exotic populations. The results of present study might have significant impact on the breeding and conservation of native chicken genetic resources in Bangladesh.

Bangladesh has a very rich genetic diversity of native chickens and one of the homelands of the Red Jungle Fowl (Gallus gallus), the ancestor of modern domestic chickens. Although, Bangladesh has a rich heritage of native poultry germplasm, recent industrialization of poultry industry is creating threat to indigenous chicken populations because farmers are reluctant to rear low producing native chickens, which strongly supported decisive measures for conserving native genetic resources.

Most Bangladeshi native chickens have a colored appearance, slow growth rate,
good taste of meat and low reproductive performance due to broodiness. They
have some morphological characters such as naked neck, frizzles, dwarfism etc.,
which have direct and/or indirect effect on tropical adaptability. The genes
that control the naked neck production traits supported better feed efficiency,
growth, carcass composition, meat yield and better tolerance to high ambient
temperature (Singh et al., 2001; Islam
and Nishibori, 2009), while The genes governing the egg production trait
in frizzles supported the increases of egg production and egg mass and reduces
mortality under hot climatic conditions (Garces et al.,
2001; Nischal and Sharma, 2002). These two major
genes are also believed to confer resistant to diseases. Therefore, the question
about the significance of local fowl as genetic pool for future breeding strategies
and as a supplier of gene complexes or single gene affecting special traits
is still open.

Genetic variability and relatedness among the native and improved breeds/lines of chicken are necessary information required because the genetic variation is considered as the primary biological resource that can be exploited in selective breeding program.

Therefore, the present study was performed to characterize the differences among Bangladeshi native chicken and imported exotic breeds/strains using RAPD markers.

MATERIALS AND METHODS

Sample collection: The sample collection and laboratory work of this
study was conducted in two phases in between July 2002 and January 2006. The
first phase of this experiment was conducted to study the efficiency of RAPD
marker for generating polymorphism in different chicken populations (Mollah
et al., 2005) and the second phase was conducted to analyze genetic
diversity among different indigenous and exotic chickens in Bangladesh. For
this experiment, five males and five females were randomly sampled from each
of the following chicken populations: Naked Neck (NN), Frizzle (FZ), Non-Descriptive
indigenous (ND), White Leghorn (WL), Rhode Island Red (RIR) and Commercial Layer
(CL) and broiler (CB) strains those were kept in the Department of Poultry Science
Farm, Bangladesh Agricultural University, Mymensingh. The characteristics of
each population are summarized in Table 1. The laboratory
work was performed in the Department of Fisheries Biology and Genetics Laboratory
and Central Laboratory of Bangladesh Agricultural University, Mymensingh.

Blood collection and genomic DNA extraction: Blood was collected and
prepared for DNA isolation by using the procedure suggested by Hoelzel
(1992). Genomic DNA was extracted from blood cell following the phenol and
chloroform method. In brief, approximately 10 μL of previously separated
blood cells were taken in a microfuge tube containing 450 μL of extraction
buffer (100 mM Tris.HCl, pH = 8.0, 10 mM EDTA and 250 mM NaCl and 1% SDS). After
adding 25 μL of proteinase K (20 mg mL-1) the mixture was incubated
at 37°C overnight for digestion. DNA was purified by successive extraction
with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol
(24:1), respectively. DNA was precipitated first using 0.6 volume of isopropanol,
peletted by centrifugation, then resuspended in TE buffer (10 mM Tris. HCl,
1 mM EDTA, pH = 8.0). DNA was reprecipitated by adding two volumes of ethanol
in the presence of 0.3 M sodium acetate and peletted by centrifugation. The
pellets were then washed with 70% ethanol, air-dried and finally resuspended
in an appropriate volume of TE buffer. DNA quality was checked by electrophoresis
in a minigel and quantified using a spectrophotometer (Spectronic Genesys, Thermo
Electron Corporation).

Primer selection: Initially, twenty decamer primers of random sequence (Kit A, Operon Technologies, Inc., Alameda, California, USA) were screened on a sub sample of two randomly chosen chickens from each population, to test their suitability for amplifying chicken RAPD that could be accurately scored. Primers were evaluated on the basis of resolution of bands, repeatability of markers and potential to differentiate populations (polymorphism). Finally, four primers (Table 2) exhibiting sufficient variability for population analysis were selected unbiasly for the analysis of the whole sample set of the seven populations.

PCR amplification and electrophoresis: The amplification conditions
were based on Williams et al. (1990) with some
modifications. The PCR reactions were carried out on each DNA sample in a 10
μL reaction mix containing 1 μL of 10x PCR buffer, 2 μL of 10
μM primer, 1 μL of 250 μM dNTPs (Takara, Japan), 1 unit of Taq
DNA polymerase (Takara, Japan) and 75 ng of genomic DNA and a suitable amount
of sterile deionized water. DNA amplification was performed in an oil-free thermal
cycler (Master Cycler Gradient, Eppendorf). Amplification profile consisted
of 3 min initial denaturation at 94°C followed by 40 cycles of 1 min denaturation
at 94°C, 1 min annealing at 34°C and 2 min extension at 72°C. After
the last cycle, a final step of 7 min at 72°C was added to allow complete
extension of all amplified fragments.

Table 1:

Description
of chicken populations used in this study

Table 2:

Parameters
of primers used in RAPD analysis

The amplified product from each sample was separated electrophoretically on
1% agarose gel containing ethidium bromide in 1xTAE buffer at constant voltage
of 120 V for 1½ h. Lamda DNA-EcoT 14 I digest and/or 100 bp ladder used
as DNA molecular size marker were run alongside the RAPD reactions on each gel.
DNA bands were visualized on UV-transilluminator and photographed by a polaroid
camera (Gel Cam Polaroid camera, Sigma-Aldrich Corp).

Genetic data analysis: The PCR amplified bands were scored visually
by two independent persons on the basis of their presence (1) or absence (0).
The scores obtained were then pooled for constructing a single data matrix,
which was used for estimating the proportion of polymorphic loci, Neis
(1973) gene diversity (h), gene flow (Nm), coefficient of gene
differentiation (GST), Neis (1978) unbiased
genetic distance (D). Significant test and construction of a UPGMA (Unweighted
Pair Group Method of Arithmetic Means) dendrogram among populations with 1000
simulated samples were carried out by using POPGENE (version 1.31) (Yeh
et al., 1999) computer program. Band sharing based intra-population
similarity indices (Si) were calculated for all possible comparisons
according to the following formula:

Similarity index (Si) = 2NAB/(NA +NB)

where, NAB is the total number of RAPD band shared by individuals
A and B. NA and NB are the numbers of fragments scored
for each individual, respectively (Lynch, 1990).

RESULTS

Out of the twenty primers four retained for RAPD analysis produced different
fragment patterns with varied number of bands. The primers yielded a total of
39 distinct bands (RAPD markers), 25 (64.10%) of which were considered as polymorphic
(either occurring in or absent in less than 95% individuals).

Table 3:

Total
number and percentage of polymorphic loci, gene diversity and intra-population
similarity indices (Si) of seven populations of chicken

The characteristics of the fragments generated by these four primers are summarized
in Table 2. Primer OPA20 produced more numerous fragments
than the other three primers. Examples of varying degree of polymorphism generated
with the four primers are shown in Fig. 1a-d.

The number of polymorphic loci, percentage of polymorphic loci, gene diversity and intra-population similarity indices of seven populations of chicken are shown in Table 3. The overall number of polymorphic loci, percentage of polymorphic loci and gene diversity were 25, 64.10% and 0.25, respectively.

The numbers of polymorphic loci were higher in native chicken than exotic ones. Among the native chickens, maximum number polymorphic loci were detected in ND (20) followed by NN (19) and FZ (18). In contrary, the degree of polymorphism was relatively low in CL and BL populations. Similarly, the within population gene diversity was relatively high (0.20-0.22) in native chickens, intermediate in RIR (0.16) and low in CL (0.13), CB (0.12) and WL (0.12) (Table 3). The coefficient of gene differentiation and the gene flow estimates considering all populations across all loci were 0.34 and 0.98, respectively.

The intra-population similarity indices (Si) were relatively high in the exotic chickens (82.45-90.03%) compared to the native ones (79.76-83.39%). Among the exotic populations, CL showed higher genetic similarity (Si = 0.900) in comparison to the RIR, CB and WL populations while among the native populations, FZ showed higher within-population genetic similarity (Si = 0.834) than the ND and NN, respectively. The lowest intra-population similarity was observed in the ND population (Si = 0.797).

UPGMA dendrogram based on Neis (1978)
unbiased genetic distance, summarizing the data on differentiation between
chicken populations, according to RAPD analysis

The overall genetic distance among native chicken was relatively low in comparison
to the exotic populations though the ND population showed somewhat higher genetic
distance with the rest two native populations. The smallest genetic distance
(0.036) was estimated between FZ and NN population pair. Among the exotic populations,
CB was separated from WL, CL and RIR populations with relatively high genetic
distance (Fig. 2) whereas the genetic distance for WL vs.
CL, RIR vs. CL, WL vs. RIR population pairs were marginally low.

Better understanding of population genetic structure is crucial to develop
a sustainable strategy for conservation and effective utilization of low productive
livestock species. In recent years, different marker systems have been developed
and applied to a range of livestock species. In this study, we used RAPD marker
to study the genetic diversity of Bangladeshi indigenous chicken and/also compared
with other exotic breeds. Although no specific markers were found to discriminate
the studied chicken populations effectively, the RAPD technique disclosed sufficient
polymorphism for population analysis. A total of 25 polymorphic loci detected
in this study indicated the effectiveness of RAPD technique to study polymorphism
and genetic relatedness among the different chicken populations. The average
number of bands obtained by individual primer in this study was ranging from
9 to 11. Since the amplification from these arbitrary sequenced primers depends
upon the presence of annealing site on template DNA, different primers are expected
to give different number of amplicons. The results agreed well with the findings
by Shivaraman et al. (2001), Ahlawat
et al. (2004) and Mollah et al. (2005).

In view of intra-population similarity indices (Si), the proportion
of polymorphic loci and Neis gene diversity (h), illustrated that the
Bangladeshi native chicken populations can be considered as genetically more
diversified than the exotic chicken populations. Relatively lower genetic variations
observed in CL and BL populations compared to native populations might be due
to differences in population structure and selection history. Since the exotic
populations have a history of long intense artificial selection for either egg
number (For example WL, RIR, CL) or body weight (CB), low amount of genetic
variability was expected in these populations. Alternatively, as the native
chicken populations are not under artificial selection pressure, each of them
is considered as large random mating population that increases the heterozygosity/genetic
variability in native chicken populations. Like the present study, Smith
et al. (1996) and Zhang et al. (2002)
have discovered higher genetic variability at genomic level in native populations
in comparison to the exotic ones.

Pair wise genetic distance and homogeneity test were performed to determine
the relatedness among different chicken populations. Lower genetic distance
among local native chickens reflected the geographical proximity between them
and the results supported the hypothesis that the geographical distance is an
important factor influencing the genetic relatedness of populations (Wright,
1943). In a survey, Shivaraman et al. (2001)
and Dehghanzadeh et al. (2009) were also observed
least genetic distance among native chickens. The higher genetic distance between
the imported CB and CL chickens indicated the remote relationship between them.
This might be due to the reason that the exotic broilers and layer chickens
were bred for different purposes and had different origin. On the other hand,
close relationship among different egg type chicken populations might be due
to their same breeding purpose. The lower observed genetic distance between
the native and exotic population pairs (FZ vs. RIR, FZ vs. WL, NN vs. RIR) indicating
that somewhat close relationship between each pair of them. The close genetic
relationship among the above native and exotic population might be due to the
efforts made over years by Bangladesh Government to improve the local chicken
populations by crossing with exotic RIR and WL chicken. Higher level of population
differentiation (GST = 0.34) and low level of gene flow (Nm=
0.98) across all loci indicated that sufficient genetic differences among different
chicken populations are present. However, to discuss the detailed population
structures, further studies are required dealing with a large number and extensively
sampled native chickens from different parts of the country and more RAPD markers.
Our future research based on integrating RAPD and microsatellite marker will
provide more details about this matter.

In conclusion, RAPD markers found sufficient nuclear DNA level variations among different chicken populations in Bangladesh. The RAPD data presented here might be a good source of information about the diversity of native chicken in Bangladesh.

ACKNOWLEDGMENT

This study was supported in part by grants from World Poultry Science Association-India Branch, under Dr. B.V. Rao WPSA (IB)/WPC96 Research Grants for Poultry Post-graduates program to M.B.R. Mollah.